West Nile virus (WNV) is a serious human pathogen with a mortality rate of 0.1%. The National Institute for Allergy and Infectious Disease (NIAID) Biodefense Research Agenda classifies WNV as a Category B Priority Pathogen on the basis of its transmission by an arthropod host, its infectivity on aerosolized exposure, and its potential threat to public health. Of particular concern is the possibility that West Nile virus might be used as an agent of bioterrorism. There is currently no vaccine for WNV available for use in humans. Thus, development of a WNV vaccine is a national biodefense and public health priority that is consistent with the overall goals of NIH and MBRS. The goals of this Pilot Research Project are to develop a candidate vaccine for West Nile virus and to test its immunogenicity in mice. This proposal incorporates two novel concepts: the use of Nodamura virus (NoV) RNA replicons to amplify WNV mRNAs in the yeast Saccharomyces cerevisiae and inoculation of animals with purified total yeast RNA containing amplified NoV-WNV RNAs. The advantages of the nodavirus expression system as a source of vaccine material include exponential amplification of the chimeric mRNA; its ability to initiate RNA replication in a wide variety of host cells, including those from mammals, insects, plants, and yeast, when the RNA is introduced by transfection; and the lack of pathogenicity of NoV for humans. Yeast cells provide a safe and inexpensive source of vaccine material, and are currently used to produce Hepatitis B vaccines. The specific aims of this proposal are: 1. Construct NoV RNA2-based replicons that contain WNV structural (glycoprotein E, gpE) or nonstructural (NS1) proteins and determine the extent to which RNA replication amplifies NoV2-gpE and NoV-NS1 mRNA and protein levels in yeast and mammalian cells 2. Evaluate the WNV structural (gpE) and nonstructural (NS1) proteins as potential vaccine candidates by defining the humoral and cell-mediated immune responses to these proteins elicited in inoculated mice. Relevance to public health: West Nile virus is a severe human pathogen that poses a threat to the public health and biosafety of the United States. Our goal is to develop safe, effective candidate vaccines that will prevent the spread of West Nile virus-associated diseases.